The long-term objective of this research is to understand how the environmental bacterium Listeria monocytogenes (Lm) adapts to life within mammalian cells to become a human pathogen. Lm is a facultative intracellular bacterium that survives as a saprophyte in soil but is capable of transitioning into a pathogen upon entry into a mammalian host. As a pathogen, Lm remains an increasingly important agent of serious food- borne infections and has been responsible for some of the largest food safety recalls in U.S. history. PrsA2 is a peptidyl-prolyl cis/trans isomerase that functions as a post-secretion chaperone or foldase and is required for Lm virulence. Preliminary data indicates that PrsA2 is uniquely required for the folding, stability, and optimal activity of secreted factors required for bacterial invasion, replication, and cell-to-cell spread within the infected host. Lm strains lacking PrsA2 function are severely attenuated in mouse models of infection, and exhibit reduced secretion and activity of at least two key Lm virulence gene products, the cholesterol-dependent cytolysin listeriolysin O (LLO) and the broad specificity phospholipase PC-PLC. Both of these gene products contribute to Lm's ability to gain access to the host cell cytosol (its required replication niche) and to spread of the bacteria into new host cells. Preliminary experiments indicate that PrsA2 is also required for the secretion and/or activity of additional Lm factors with roles in bacterial virulence. This proposal seeks to elucidate the role of PrsA2 in regulating the activity of Lm secreted proteins used to secure the bacterium's intracellular replication niche. Aim 1 will undertake a molecular analysis of PrsA2's role in promoting the secretion and activity of LLO and PC-PLC. Aim 2 is designed to elucidate PrsA2 interacting partners using genetic analysis. Aim 3 experiments focus on the functional analysis of the role of PrsA2 in Lm secretion and bacterial virulence. The ultimate goal of the specific aims will be to elucidate how PrsA2 modulates the secretion and/or activity of virulence factors to promote bacterial pathogenesis in mammalian hosts. PUBLIC HEALTH RELEVANCE: Listeria monocytogenes represents an increasingly significant health threat as it has been associated with several multi-state food-borne outbreaks that have resulted in thousands of illnesses and several hundred deaths within the past few years. The largest and most expensive (>$140 million) recalls of food products in history continue to occur as a result of L. monocytogenes contamination, and the bacterium generally ranks as the third or fourth most common cause of bacterial meningitis in North America. This proposal will elucidate how L. monocytogenes regulates the activity of secreted factors critical for bacterial virulence;this Information may result in the identification of new bacterial drug targets as well as enhance the development of L. monocytogenes-based vaccines for delivery of foreign antigens.